Project Summary Synapses are the essential functional unit of the nervous system. Formation of synapses requires the establishment of highly specialized cell-cell junctions, a process orchestrated by complex extracellular signaling networks. The synaptic cytoskeleton is a major target and effector of these signaling networks, and our understanding of the precise roles of the cytoskeleton, particularly of microtubules (MTs), is rapidly evolving. The importance of several MT associated proteins (MAPs), including Futsch, VAP-33, and spastin, to synapse development have recently been identified. Our work has furthermore identified the MAP TACC (Transforming acidic coiled coil), which is best known for its roles in regulating the mitotic spindle through recruitment of the MT polymerase Msps, as novel negative regulator of synaptic growth. We first identified TACC as a suppressor of CLASP, a MT effector downstream of Abelson (Abl) kinase. Both CLASP and TACC belong to the specialized class of MAPs known as plus tip tracking proteins (+TIPs). Functional screens of the CLASP interaction network in our lab showed that TACC regulates ex vivo MT dynamics as well as the growth of the Drosophila neuromuscular junction (NMJ) in vivo. We furthermore found that TACC localizes to the active zone (AZ) and controls AZ size, consistent with the interactions of TACC with AZ components. We hypothesized that TACC may regulate synaptic MTs to control the formation and function of the AZ, ultimately affecting overall synaptic growth and function. We propose to perform rigorous characterization of TACC loss- of-function (LOF) phenotypes throughout development, as well as to further characterize its regulation of AZ size and its interactions with known AZ/peri-AZ components (Aim 1). We will test precise effects of TACC on regulating synaptic MT dynamics (Aim 2). Specifically, we will determine if TACC regulates MT dynamics by developing a novel imaging and analysis platform (in collaboration with DRVision) for in vivo tracking of EB1- GFP labeled dynamic MTs. We will also test the effect of TACC in MT structure/organization, and determine if TACC regulates MT-based axonal transport. In addition, we will test the interactions of TACC with CLASP and Msps, which are confirmed partners of TACC in other contexts, as well as with the key AZ regulator and scaffold Liprin-a, to understand the role of TACC within a broader functional pathway (Aim 3). Through this work, we will determine the interactions of MTs with the AZ and their biological significance. We will also uncover new roles for +TIPs, which were not previously known to function in synapse development.